The further characterization and study of 3 important enzymes that are targets for chemotherapeutic agents is proposed. By using techniques of recombinant DNA technology, we propose to generate sufficient amounts of two of these enzymes (dihydrofolate reductase and thymidylate synthase) and altered forms of these enzymes for detailed studies of structure, characterization, and interaction with inhibitors. The regulation of thymidylate synthetase will be further studied. Based on this and other information, new approaches to drug development, in particular inhibition of polyglutamate synthetase are planned. Strategies for prevention and eradication of drug resistant cells, based on concurrent or alternating therapy with folate contagonists have been formulated, and will be tested using tumor cells propagated in vitro, and with murine tumors propagated in vivo.